Topic 7 Recomb DNA repair

Question 1

What are the purposes of homologous recombination

Answer 1

Recombine To create new genetic diversity by exchanging genetic information between two dna molecules with similar sequences

Recombination repair To fix singles strand and double strand breaks

Question 2

How is meiosis both helpful and not helpful

Answer 2

Meiosis gives genetic exchange and diversity

but to do this chromosome arms need to break which is Dangerous because if the breakage doesn’t properly repair, loss of dna info and then mutations can happen

Question 3

How do DSB arise

Answer 3

During DNA replication when the fork encounters a ss break in the template strand (now goes into the daughter strand)

Meiotic recombination

Exposure to UV light or gamma radiation

Oxidative DNA damage during respiration

Question 4

Explain what scenarios happen when there is a damaged template strand during replication

Answer 4

1. Translesion synthesis: just read and moves past the lesions
2. The replication fork stalls at the lesion causing fork regression
3. If a full single stranded break in the template, fork collapses, and a double stranded break is formed with the template strand (other strand is normal length)
4. The lesion is bypassed/jumped over, which leaves a single stranded gap in the daughter strand, replication continues downstream

Question 5

Expo n what happens right after a double stranded dna break happens (in one chormosomes)

Answer 5

Exonucleases make 3’ overhangs by chewing the 5’ ends of the broken dna stands

DNA recombinase causes one 3’ overhang strand to invade and recombine with the homologous chromosome, making a D-loop structure (due to displacement of the homologous strand during invasion)

Double crossover: The D-loop migrates/expands and allows the other end of the broken strand to also invade

The bottom broken strand uses the bottom homologous strand as a template and the top broken strand uses the top homologous stand as a template

The 3 prime overhangs are used as primers for dna pol to extend and repair the broken strands using the homologous strands as a template

Question 6

What is used as a primer in DSBR to extend the broken strands

Answer 6

The 3’ overhangs are used as primers for extension

Question 7

What are the two ways to complete DSB repair

Answer 7

SDSA (synthesis dependent strand annealing) pathway

DSB repair pathway

Question 8

What is SDSA (synthesis dependent strand annealing) pathway

Answer 8

After using the homologous chromosome as a template, the invading strands dissociates and anneal to each other and ligate

No crossing over event, just repairing the lesions using the homologous as a template

Question 9

What is DSB repair pathway

Answer 9

The invading strand is linked in the d loop structure

This makes 2 Holliday junctions/intermediates that are fixed by Holliday intermediate resolvases

Can be resolved in two ways

Question 10

What are the two ways to resolve the Holliday junctions and the out comes

Answer 10

X x X resolution: non crossover

X x Y resolution: cross over and exhange of genetic info

Question 11

Explain what happens during a fork collapse

Answer 11

When there is a break in the template strand in the replication fork

The lagging (top) strand keeps going but the leading strand stops at the break and the arm with the break detaches

causing a DS dna break and The fork collapses

the repair needs a reattachment of the broken arm to recreate the replication fork

Question 12

Explain how recombinational DNA repair fixes a collapsed fork

Answer 12

Have the bottom normal chromosme and the dS break top chromsome

Exonucleases remove part of the 5’ end of the broken strand (so cleave the 3-5 stand) to make a 3’ overhang on the bottom strand (5-3 strand)

Recombinase binds to the 3’ overhang to promote strand invasion and make a Holliday junction

The 3’ overhang pairs with its complimentary strand on the homologous chromsome

Then branch migration occurs to let the strand invade further

Fork is remade when the Holliday intermediate is resolved followed by ligation

Question 13

Whag is branch migration

Answer 13

The movement of a branch point in the branched DNA formed by two DNA molcules with identical sequences

This can make a Holliday junctions but The net amount of duplex dna does not change

Question 14

Explain what fork regression is

Answer 14

When a lesion is encountered in the replication fork, the fork starts to move backward

The fork was open then starts sealing back up

This lets the lesion stay reannealed with the template strand (not replicated since not in the fork)

Question 15

Explain the steps of fork regression

Answer 15

Fork Encounter lesion

The replicated strands in the fork go in the opposite direction (backward) so the fork stays closed

NER can repair the lesion and restart replication, the extra regressed replicated strands (short dna arm) get digested

Or

No NER, the short dna arm replicates using the other regressed strand as a template

Then the replication fork opens more to let the newly synthesized strand pair with the lesion (not perfect pairing) so replication can restart

The lesion can be repaired later

Question 16

Overall What does fork regression do

Answer 16

Restarts replication

Question 17

When is dna replicated

Answer 17

Before meiosis

Question 18

Whag are the stages of prophase 1 in meiosis and what do they mean

Answer 18

Leptotene: the chromosomal condensation happens

Zygotene: the homologous chromosomes cross over through synapsis making the synaptonemal complex to allow recombination

The synapsed chromosomes make a bivalent/tetrad

Pachytene: synapsis is conpleted

Diplotene: synaptionemal complex disappears and homologous chromosomes start to move apart

Question 19

What are chiasmata

Answer 19

Remaining points of attachment between homologous chromosomes during crossover/exchange

Question 20

Which histone can you use to scan the chismata during exchange

Answer 20

When dna is breaking during recombinations

you can see h2A-X because it replaces H2A at the site of double stranded breaks

Question 21

What needs to happen for meiotic recombinations to happen

Answer 21

double stranded breaks in The chromsome

Question 22

What catalyzes double stranded breaks during recombination and how

Answer 22

Spo11 in S.Cerevisiae

Uses its tyr residues (has oh) to act as a nucleophile in a transesterification reaction

This made the DSB + spo11 with spo11 linked to the DSB by a 5’ phosphotyrosyl linkage

The are 2 spo11 one on top strand one on bottom strand

Question 23

What is spo11 related to

Answer 23

It is closely related to the Topo II in eukaryotes

Question 24

Explain what happens after spo11 male the DSB to prepare for recombinations

Answer 24

The Mre11-Rad50-Xrs2 endonuclease complex binds to each spo11 and cleaves the dna on the 3’ side of each spo11 to release the 5’ ending dna fragment with spo 11 attached

This makes 3’ overhangs

Nuclease Sae2 degrades the 5’ end of cleaved strand more to enhance the 3’ overhangs

Sgs2-Dna2/exo1 complex further degrades the 5’ ends to make even longer overhangs

Then RPA is loaded into the 3’ overhangs to keep the dna single stranded and RecA recombinases are allow for strand invasion for recombination

Question 25

What are the two possible fates of DSB in meiosis

Answer 25

Crossover or non crossover

But both have gene conversion

Question 26

What is gene conversion

Answer 26

The non reciprocal transfer of genetic info as a result of DNA repair especially during meiosis

Question 27

How can recombination happen during mitosis

Does it happen often

Answer 27

Rare

Caused by Exposure to ionizing radiation, endonucleolytic action, a replication fork with a template strand break

Question 28

When does mitotic recombination happen

What happens after the DSB happens

Answer 28

Not during mitosis

During the S and G2 phases of cell cycle

The DSB triggers a checkpoint that halts the progression of the cell cycle to allow repair to occur

Question 29

How does mitotic DSB repair happen

Answer 29

Similar to meiotic recombination

Happens like this:

A terminally differentiated cell has a Mitotic DSB

Nuclease Make 3’ overhangs

RPA coats the single stranded dna to keep single stranded

Rad52 and Brca2 recombination mediator protiens come

Rad52 recruits Rad51 and they both start recombinations, rad51 triggers the first strand invasion and rad 52 triggers the second

Leads to either SDSA or DSBR

Question 30

Other than using DSB to recombine and repair what do DSB help with

Answer 30

Switching between different mating types

Question 31

What two mating types does the haploid state of S .cerevisiae have

Answer 31

The S cerevisiae can be in diploid or haploid state

Would prefer diploid state but when conditions are not good they go into haploid state

In haploid state: a or alpha (like male or female)

The genes in the MAT locus in their chromosome determines whether they are a or alpha

Question 32

Explain how the mating types determination in haploid S cerevisiae works

Answer 32

Have the chromosme with
HMLaplha, mat locus, and HMRa

The genetic info for MAT alpha is stored but not expressed in HMLaplha

The genetic info for MATa is stored but not expressed in HMRa

The only time aplha or a is expressed is if those MAT genes are in the MAT locus

Question 33

Explain how recombinational repair of DSB switches the mating type in haploid S cerevisiae

Answer 33

50/50 chance of cells expressing a or aplha mating types, meaning they switch across generations

The nuclease HO cleaves the mat locus to make a DSB

The free ends are processed to make 3’ overhangs

RAD51 binds to the overhangs to do strand invasion into the new mating type chromosme

Nucleases remove the original mating type info

DNA pol extends the invading 3’ end using the new mating type chromosme as a template

Depending on what mating type was expressed in the mat locus before the break, the opposite type will come in to fix the break through gene conversion

Ex. MATa broke, recombines with HMLaplha comes, now MATalpha

This is done by SDSA repair

Question 34

How can DSB be repaired if the homologous template strands aren’t available

Answer 34

Non homologous ends joining (NHEJ)

Question 35

When does NHEJ happen

Answer 35

The differentiated mammalian cells that rarely divide spend most of their time in the G1 and G0 phases, they use NHEJ to repair their dna damages

But NHEJ doesn’t not conserve the original DNA sequences meaning it can introduce mutations

Question 36

Explain how NHEJ happens

Answer 36

The DSB happens in G1 or G0

The ku70 and ku80 heterodimer binds to the broken ends of the DNA strands and act as molecular scaffolds

They recruit DNA-PK (kinase) and the nuclease Artemis

This ku/DNAPK/Atermis complex hold together (synapse) the broken ends of the DNA

helicase separates the DNA which activates the DNA PK

DNAPK phosphorylates Artemis to activate its endonuclease activity

Artemis cleaves the DNA segments to turn the previous blunt ends into sticky ends

Then the strands are annealed, the small DNA gaps are filled by polymerases

The nicks are sealed by a protien complex called XRCC4, XLF, and DNA ligase IV

Question 37

What causes there to be more mutations in NHEJ

Answer 37

Since the Artemis endonuclease cleaves the blurb ends to make sticky ends then these ends are just stuck together

some dna info is lost when this happens and sequence is lost and mutation can happen

But choose between survival and death

Question 38

What are XRCC4 and XLF

Answer 38

X ray cross complementation group

XRCC4 like factor

Question 39

How can DNA repair systems be used for gene editing

Answer 39

In CRISPR, we can knock in or knock out a tag

HOMOLOGOUS RECOMBINATION: To knock in this tag (ex GFP) we can use a designed donor dna with the tag to recombine with the target dna that has a DSB from the CAS9 from the crispr

NHEJ: to knock out a gene (make mutations) the DSB in the DNA from Cas9 can undergo NHEJ since no template. A mutation is introduced and the gene is non functional